In many assays, it is desired to measure the concentration of two or more analytes simultaneously in one sample. This is also the case in affinity-based assays, such as immunoassays. Often, the analytes are present in very different concentrations. This may be a problem for affinity-based assays when the concentration of the different analytes are to be measured simultaneously, since there is a risk that the signal resulting from the measurement of one analyte may saturate or affect the signal resulting from the other.
Saturation may occur in an affinity-based assay comprising capturing means for instance when all capturing sites are occupied. Saturation may also occur when a transducer, such as a sensor measuring fluorescence from labelled molecules, is saturated.
In the prior art there has been made efforts to solve the problem to measure two or more analytes with very different concentrations in affinity-based assays. One approach is to split the sample into several aliquots, which are diluted to different concentrations. Another approach is to adjust the gain of the transducer, detector or sensor to the different concentrations.
U.S. Pat. No. 7,271,009 discloses immunological assays for several biological markers in a sample comprising the use of particles. Each particle is coated with one type of molecules taking part in the affinity-based assay. There are several types of particles, each coated with one type of molecule. To handle situations where the levels of the various analytes differ considerably, the signal is lowered for some of the analytes by the use of a diluting agent. It is described that the diluting agent does not engage in the specific binding with any of the analytes. The diluting agent competes for the sites available on the particle and lowers the coating density of the analyte. The number of analyte molecules which are captured are thus reduced by using the diluent. There is also described an embodiment where some particles are coated with an agent which increases the sensitivity for a particular analyte.
In some analysis devices according to the state of the art, a capturing antibody or a capturing molecule captures one or more analytes. The binding of analytes to the capturing sites is interrupted before equilibrium before all capturing sites are saturated so that the signal shall not become too high.
In some situations there is only one binding epitope available on at least one the analytes to be detected.
Although techniques according to the prior art are used, there is room for an improvement regarding that the sample may have to be diluted in several steps in several aliquots and/or that the gain of the transducer, detector and/or sensor has to be adjusted to accommodate different concentration levels. It is also desired to have a quick assay with as few steps as possible, where for instance particles do not have to be handled. It is also desirable to have an assay in which no diluent has to be added. An assay where it is possible to measure at least one analyte with only one epitope is also desired. It is also desired to have an assay which is able to measure the concentration of an analyte with only one epitope in a liquid sample mixture.